Streptococcus pneumoniae (pneumococcus) is an extremely serious human respiratory pathogen that kills well over two million people annually worldwide. Multidrug resistance is increasing in S. pneumoniae clinical isolates at an alarming rate. Many clinically relevant antibiotics, including -lactams and vancomycin, target peptidoglycan (PG) biosynthesis. PG forms the major rigid structure in the cell wall that determines cell shape and size and serves as the scaffolding onto which other pneumococcal virulence factors are covalently attached, including capsule, teichoic acids, and sortase-transferred proteins. Despite its importance to physiology and pathogenesis, little is known about the supramolecular protein complexes that mediate PG biosynthesis on the cell surface of S. pneumoniae and other ellipsoid-shaped ovococcus Gram-positive pathogens. The long-term goal of this project is to fill in this major knowledge gap about the locations, interactions, regulatory dynamics, and functions of the supramolecular protein complexes that mediate pneumococcal PG biosynthesis. This proposal is based on a large body of new papers and unpublished data that demonstrates numerous unique and unexpected properties of PG biosynthesis in S. pneumoniae. PG biosynthesis is a broad topic that encompasses both PG synthesis and PG remodeling by hydrolysis. This five- year proposal consists of four synergistic Specific Aims that address some of the most important outstanding problems in ovococcus PG biosynthesis. These four Aims are conceptually linked to the central hypothesis that PG synthesis and PG remodeling enzymes function as dynamic supramolecular complexes, whose activities and interactions are choreographed with each other and with cell division. These Aims were chosen, because they will yield fundamental principles about PG biosynthesis, are supported by strong new data, are experimentally tractable, feed into each other, and will have high impact on the field. Related Aims 1 and 2 will elucidate how penicillin binding proteins (PBPs) are localized, activated, and tied to stages of cell division through interactions with a small number of essential master organizer proteins. Aim 3 will test the hypothesis that PG hydrolysis involved in PG remodeling is coupled directly to cell division. Aim 4 will determine how PG hydrolases modulate the supply of PG pentapeptide substrates used by PBPs and whether PG peptides play roles in organizing PG synthesis. A comprehensive strategy that combines results from innovative genetic, biochemical, cell biology, and microscopic approaches with those from colonization and infection models will be used to meet these Aims for this primary bacterial pathogen. Results from this proposal will challenge and expand paradigms and models about PG biosynthesis in S. pneumoniae and other ovococcus pathogens. Since the cell surface is critical to pneumococcal virulence and extracytoplasmic proteins are accessible and druggable, there is an expectation that some of the critical PG synthesis and remodeling proteins studied in this proposal will emerge as new antibiotic and vaccine candidates.